In vitro studies on the role of the blood-brain barrier (BBB) in neuroimmunological disorders involving migration of cells across the BBB have been hampered by the inability to establish long-term cultures of cerebral vascular endothelial cells (EC), the main component of the BBB. Using hybridoma technology, we have established a panel of hybridomas (fusion of Lewis rat and mouse myeloma NS-1 cells) which secrete antibodies which recognize EC. Characterization of these antibodies by immunofluorescence microscopy, FACS analysis and ELISA techniques demonstrate antibodies which react with only murine cerebral vascular EC, with EC isolated from other than of the mice, or react with EC from other species. These antibodies were also characterized according to their type (IgG vs IgM). The results demonstrate the isolation of pure monoclonal antibodies to CNS-EC which will be useful to further progress on establishment of long-term EC cultures and other studies utilizing cerebral vascular EC.